Abstract
The membrane lipids diacylglycerol (DAG) and phosphatidic acid (PA) are important second messengers that can regulate membrane transport by recruiting proteins to the membrane and by altering biophysical membrane properties. DAG and PA are involved in the transport from the Golgi apparatus to endosomes, and we have here investigated whether changes in these lipids might be important for regulation of transport to the Golgi using the protein toxin ricin. Modulation of DAG and PA levels using DAG kinase (DGK) and phospholipase D (PLD) inhibitors gave a strong increase in retrograde ricin transport, but had little impact on ricin recycling or degradation. Inhibitor treatment strongly affected the endosome morphology, increasing endosomal tubulation and size. Furthermore, ricin was present in these tubular structures together with proteins known to regulate retrograde transport. Using siRNA to knock down different isoforms of PLD and DGK, we found that several isoforms of PLD and DGK are involved in regulating ricin transport to the Golgi. Finally, by performing lipidomic analysis we found that the DGK inhibitor gave a weak, but expected, increase in DAG levels, while the PLD inhibitor gave a strong and unexpected increase in DAG levels, showing that it is important to perform lipidomic analysis when using inhibitors of lipid metabolism.
Highlights
The cell itself and all of its organelles are surrounded by a lipid bilayer, made up of phospholipids, sphingolipids and sterols
Since RI and CAY94 had a tendency for higher effect on ricin sulfation compared to RII and CAY93, respectively, we decided to focus on the DAG kinase (DGK) inhibitor RI and the phospholipase D (PLD) inhibitor CAY94 for the rest of the study
Gene expression was analyzed by qPCR and we found that HEp-2 cells have high expression of PLD3 and lower expression of PLD1 and PLD2 (Fig S7), which is similar to the expression pattern of PLDs in HeLa cells
Summary
The cell itself and all of its organelles are surrounded by a lipid bilayer, made up of phospholipids, sphingolipids and sterols. It is becoming increasingly clear that lipids play an active role in numerous cellular processes, including transport events and signaling Both diacylglycerol (DAG) and phosphatidic acid (PA) are important membrane lipids and second messengers that contribute to cellular processes either by their biophysical effect on the membrane or by recruiting proteins to the membrane. Due to their small head groups, DAG and PA have conical shapes, and local accumulation of DAG or PA can impart negative curvature to membranes thereby facilitating membrane budding, and fusionand fission reactions during vesicular transport [1]. Number of small Cell area arge and giant endosomes endosomes
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